This invention relates generally to dietary energy supplements, and, in particular, to a novel method and composition beneficial to functioning of the heart, skeletal muscles and other tissues of humans and other mammals with carbohydrate energy forms during exercise stresses and subsequent recovery.
The present invention takes advantage of discoveries of the classic (cell-cell, organ-organ) xe2x80x9cLactate Shuttle,xe2x80x9d and the xe2x80x9cIntracellular Lactate Shuttlexe2x80x9d mechanisms by Brooks (1984, 1998). The xe2x80x9cLactate Shuttle Hypothesisxe2x80x9d holds that lactate plays a key role in the distribution of carbohydrate potential energy which occurs among various tissue and cellular compartments such as between: cytosol and mitochondria, muscle and blood, blood and muscle, active and inactive muscles, white and red muscles, blood and heart, arterial blood and liver, liver and other tissues such as exercising muscle, intestine and portal blood, portal blood and liver, zones of the liver, skin and blood, and astrocytes and neurons in the brain. Studies on resting and exercising humans indicate that most lactate (70-80%) is disposed of through oxidation, with much of the remainder converted to glucose and glycogen. Studies on canine muscles made to contract in situ also yield the result that lactate is rapidly oxidized (Gladden et al., Zinker et al.). Lactate transport across cellular membranes occurs by means of facilitated exchange along pH and concentration gradients (Roth and Brooks 1990a, 1990b) involving a family of lactate transport proteins now called monocarboxylate transporters (MCT""s) (Garcia et al., 1994; Price et al., 1998). Current evidence is that muscle and other cell membrane lactate transporters are abundant with characteristics of high Km and Vmax. There appears to be long-term plasticity in the number of cell membrane transporters, but short-term regulation by allosteric modulation or phosphorylation is not known to occur.
The key to recognition of an xe2x80x9cIntracellular Lactate Shuttlexe2x80x9d is recognizing that in addition to cell membranes, mitochondria also contain monocarboxylate transporters (mitochondial MCT""s or mMCT""s) and lactic dehydrogenase (mLDH). Mitochondrial MCT""s exist in the mitochondrial inner membrane, and possibly also the outer membrane (FIG. 1), although presence of an outer mitochondrial membrane MCT is not essential because it is highly permeable. The Intracellular Lactate Shuttle also requires presence of mitochondrial lactate dehydrogenase (mLDH) located on the inner membrane and in the intra-membrane (periplasmic) mitochondrial space. mLDH is necessary to convert lactate, the predominant plasma and intracellular monocarboxylate, to pyruvate, for transport via mMCT into the mitochondrial matrix for catalysis by pyruvate dehydrogenase (PDH) and entry into the tricarboxylic acid (TCA) cycle. Therefore, mitochondrial monocarboxylate uptake and oxidation, rather than translocation of transporters to the cell surfaces, regulate lactate flux in vivo. Key discoveries in basic science are that lactate enters mitochondria, but that pyruvate is oxidized in the mitochondrial matrix.
A. Use of Glycerol-lactate Esters for the Cardiac and Skeletal Muscle Energy
Providing energy sufficient to optimize performance is extremely important for hearts and skeletal muscles under stress of work load. Resting healthy hearts rely on exogenous, blood borne free fatty acids (FFA) as their main energy source with carbohydrate (CHO) derived fuel sources comprised of glucose and lactate playing secondary roles. For instance, in a resting person FFA may provide 80% of energy, glucose 5%, and lactate 15% (Gertz et al., 1988; Wisneski et al., 1987). However, under exercise and other stresses total energy demand increases and the fuel mix changes with the contribution of FFA falling to 40%, glucose use increasing absolutely but remaining at about 5%, and lactate the remainder (55%). During rest lactate is relegated to a minor role as an energy substrate for the heart because arterial lactate concentration is low (xe2x89xa61.0 mM). However, during physical exercise lactate predominates as the cardiac fuel energy source because production in working muscle and other tissues causes blood lactate concentration to rise to a level (2-20 mM) sufficient to be taken up and oxidized within the heart. As indicated in FIG. 1, exogenous lactate gains entry to cardiocytes because of cell membrane lactate transporters. Those transporters facilitate lactate flux down concentration and hydrogen ion (H+) gradients. Within cardiocytes, lactate gains entry to mitochondria via another lactate transporter pool, also along concentration and H+ gradients.
Taking advantage of new knowledge of the role of lactate in cardiac and skeletal muscle metabolism, Kline et al. studied performance and efficiency of hearts removed from rabbits after hemorrhagic shock. When concentrated sodium lactate was added to the isolated working hearts taken from shocked animals, performance was significantly enhanced. This practical demonstration of the use of lactate as a fuel and anaplerotic substrate fort the TCA Cycle in hearts did not address the problem of the sodium load and its consequences imposed from either oral or intravenous administration of concentrated salt solutions.
Realizing that CHO-derived energy sources increase cardiac performance, some investigators have attempted to promote cardiac energy resuscitation after ischaemic attacks by providing glucose, sometimes with insulin and potassium. Currently used cardioplegic solutions containing glucose, insulin and potassium are sometimes referred to as GIK. Other investigators have attempted to provide pyruvate. However, from the physiological perspective such attempts are less than optimal, or misguided, because lactate, not glucose or pyruvate, is the major fuel for the heart under stress.
Recently, results of clinical trials (Ceremuzynski et al., 1999) have not confirmed viability of systemically administered GIK in the management of cardiac episodes. While GIK solutions do positively influence performance of stunned isolated hearts perfused and bathed in artificial solutions, unless GIK is administered into coronary arteries, significant effects on either cardiac performance or survival of ischaemic episodes including MI is not to be expected (Apstein and Opie, 1999). Simply, GIK can not be expected to have much effect because glucose is never the major fuel for the heart. The better approach is to provide lactate in a form that can benefit cardiac metabolism.
U.S. Pat. No. 5,294,641, herein incorporated by reference, is directed to the use of pyruvate to prevent the adverse effects of ischemia in heart muscle. The pyruvate is administered prior to a surgical procedure to increase a patient""s cardiac output and heart stroke volume. The pyruvate is administered as a calcium or sodium salt. The pyruvate can alternatively be an ester of pyruvate acid such as ethylamino pyruvate. Pyruvate is used because it is a cellular energy source; but while providing exogenous pyruvate may be potentially efficacious for heart muscle, practically the applicability is limited (vide infra).
With due consideration to growing acceptance of pyruvate as an effective component of reperfusion solution, it has been recognized that traditional pharmacological pyruvate compounds, such as salts of pyruvic acid, are not particularly physiologically suitable. For example, inorganic salts of pyruvate lead to the accumulation of large concentrations of inorganic ions (e.g., potassium, calcium or sodium) in body fluids. Accordingly, while potentially suitable to organ preservation, the salt-pyruvate compounds are not ideally suited to treating an organ or supplementing energy in an active person in vivo, and it is recognized that a need exists to deliver a monocarboxylate (pyruvate-like) compound with is more physiologically appropriate.
In this regard, U.S. Pat. No. 5,283,260, herein incorporated by reference, is directed to treatment of diabetes with a physiologically acceptable form of pyruvate. The patent discloses a pyruvate compound in the form of a covalently linked pyruvate-amino acid. By utilizing this type of pyruvate delivery system, the negative effects of inorganic-pyruvate salts are avoided. However, administration of large amounts of pyruvate-amino acid compounds may result in an amino acid nitrogen overload which could harm patients with liver and/or kidney pathology.
Similarly, U.S. Pat. No. 5,667,962, herein incorporated by reference, is directed to use of pyruvate thiolester for the prevention of cardiac reperfusion injury. The intention of that invention is to provide a compound comprising covalently linked pyruvate and N-acetylcysteine. However, the design of the material is flawed in its purpose and mode of action.
Not withstanding use of compounds of complexes of pyruvate and pyruvate-compounds in cardioplegia and organ transplantation procedures, as well as covalently linked compounds involving mixtures of pyruvate and amino acids with antioxidant characteristics such as embodied in the above-identified U.S. patents, the emphasis on pyruvate as a monocarboxylate to deliver to stressed organs and tissues is misplaced. In fact, any attempts to utilize pyruvate as an agent to improve the status of working heart and skeletal muscles results in a delayed response because lactate, not pyruvate, is the preferred compound exchanged (xe2x80x9cshuttledxe2x80x9d) among organs, tissues, cells, and intracellular compartments. Tissue levels of lactate exceed those of pyruvate by 10 to 100-fold, and cell membrane monocarboxylate transporters are specific to lactate, not pyruvate. Beneficial effects of pyruvate administration accrue only after conversion to lactate, which is the preferred material for cell-cell exchange via the xe2x80x9cLactate Shuttle.xe2x80x9d
As stated by Sumegi et al. (p. 77) who utilized nuclear magnetic resonance spectroscopy (NMR) and [3-13C]pyruvate tracer to study pyruvate metabolism in hearts of living rats: xe2x80x9cThe infused [3-13C]pyruvate was quickly converted to [3-13C]lactate in the blood of Wistar rats.xe2x80x9d [NB, this pyruvate to lactate conversion is due to presence of lactate dehydrogenase (LDH), an enzyme highly abundant in erythrocytes, such that in blood the lactate/pyruvate ratio is normally 10 and can increase an order of magnitude under physiological stress (Brooks, 1998).] Surprised by their results and unable to explain them, with some trepidation Sumegi et al. went on to state (p. 80): xe2x80x9cThese data show that the extracellular lactate is preferentially taken up by a portion of cytoplasm which converts lactate to pyruvate and transfers it to the mitochondrial reticular network.xe2x80x9d However, in making the statement concerning conversion of lactate to pyruvate in cytoplasm, Sumegi et al. recognized a major problem in interpretation of their data. By failing to recognize the existence of a mitochondrial form of lactic dehydrogenase (mLDH, FIG. 1), they had to xe2x80x9cassume that a fraction of the cytoplasm associated with the mitochondrial reticular network is specialized for converting the lactate to pyruvate, with the pyruvate being channeled to the mitochondria.xe2x80x9d [NB, in striated muscle (i.e., heart and skeletal) mitochondria do not exist as discrete organelles, but as part of a large, interconnected network, the Mitochondrial Reticulum (Kirkwood, et al.)]. As indicated by presence of mLDH (FIG. 2), the highly improbable assumption of Sumegi et al. is unnecessary, and the same physiological result is readily accomplished because of LDH.
Paradoxically, the addition of exogenous lactate to the blood of mammals has alkalinizing effects because lactate removal from the blood, whether by oxidation or gluconeogenesis, requires a proton (in the ratio of 1:1, protion:lactate anion) for transport and metabolism. Thus, by virtue of the acid/base chemistry in mammals, addition of lactate anion to plasma mitigates the presence of lactic acidosis.
(1) Data of Cell Membrane Lactate Uptake Taken from Roth and Brooks (1990a, 1990b)
Sarcolemmal vesicles were isolated from rat skeletal muscle and effects of various monocarboxylates including L(+) and D(xe2x88x92) lactate (FIG. 2), and other monocarboxylates were determined (Roth and Brooks, 1990a, 1990b). Results indicate saturation kinetics and stereospecificity for the L(+) compared to the D(xe2x88x92) isomer of lactate.
These and other characteristics (e.g., pH dependency, temperature sensitivity and inhibition by known monocarboxylate inhibitors such as CINN, vide infra) indicate presence of a sarcolemmal lactate transport protein. Further, results indicate far greater affinity for lactate (FIG. 2), than for pyruvate (FIG. 3).
(2) Data of Mitochondrial Lactate Uptake and Oxidation Taken from Brooks et al., 1990a
(a) Inhibition of Mitochondrial Lactate and Pyruvate Uptake and Oxidation by CINN: Traditionally, several substrates, and combinations of substrates have been used to study mitochondrial respiration in vitro. Pyruvate-malate has usually been used to probe mitochondrial Complex I, succinate Complex II, and TMPD+ascorbate Complex III. In contrast, lactate or lactate-malate has been infrequently used. However, pyruvate and lactate are known to share the sarcolemmal lactate transporter(s), and pyruvate gains access to the mitochondrial matrix by means of facilitated transport. Oxidation of lactate by isolated mitochondria is permitted by the presence of a mitochondrial pool of LDH which provides matrix pyruvate from exogenous lactate. To establish that lactate gains access to the mitochondrial matrix via facilitated exchange via a monocarboxylate (ACT) transport protein, we utilized polarography and inhibition by the known MCT inhibitor xcex1-cyano-4-hydroxycinnamate (CINN). Results on rat liver mitochondria are shown in FIG. 4.
Results show CINN inhibition of pyruvate and lactate oxidation, but bypass of the CINN block by succinate, which gains access to the matrix by a different transport mechanism and which donates electrons to Complex II, in contrast to lactate and pyruvate which are NADH-linked substrates and donate electrons to Complex I. Additionally, results of experiments on rat liver mitochondria with 10 mM glutamate as substrate show no measurable effect of CINN on states 3 or 4 respiratory rate, RCR and ADP/O (data not shown). Absence of an effect of CINN on glutamate oxidation by isolated mitochondria is of value because, like pyruvate, glutamate is an NADH-linked substrate. Thus, the effect of CINN on pyruvate and lactate oxidation is upstream of Complex I.
(b): Presence of MCT1 or a MCT1 Homologue in Mitochondria: Mitochondria were isolated from skeletal muscle, rat liver and heart by standard techniques of cell fractionation. Subsequently, skeletal muscle mitochondria were probed with a polyclonal antibody to the C-terminus of rat MCT1 (Nxe2x80x2-CPLQNSSGDPAEEESPV-Cxe2x80x2), and results of a Western blot analysis displayed in FIG. 5. The results indicate presence of a mitochondrial protein which reacts with an antibody to the C-terminus of MCT1. To exclude the possibility of contamination from sarcolemmal MCT1 in the mitochondrial preparation, mitochondrial and cell membrane fractions were probed with antibodies to MCT1 and the cell membrane Glucose Transport Protein #1 (GLUT1). Scarcely detectable levels of GLUT1 in mitochondrial indicate minimal contamination from cell membranes in the mitochondrial preparation. Thus, it is evident that rat striated muscle mitochondria contain a monocarboxylate transporter with high homology to MCT1. Further, similar results have been obtained on human skeletal muscle and muscle mitochondria (Dubouchaud et al.).
(c): Presence of Lactic Mitochondrial Dehydrogenase (mLDH): Mitochondria were isolated from rat liver and heart by standard techniques of cell fractionation. Subsequently, mitochondria were treated by gel electrophoresis and the results displayed in FIG. 6. The results indicate presence of mitochondrial LDH, which is mainly of the H4 isoenzyme in heart and red skeletal muscle (not shown). In contrast, liver mitochondria contain only the LDH5 isoform, whereas both LDH4 and LDH5 are present in cytosol of rat liver. These results support the conclusion of separate cytosolic and mitochondrial pools of LDH in rat muscle, liver and heart. Again, the presence of LDH in human muscle mitochondria has been demonstrated (Dubouchaud et al.).
Accordingly, it is desirable to have an alternative physiologically compatible therapeutic compound based on lactate, not pyruvate for lactate is the monocarboxylate selected by nature for exchange in the blood and between and among cells, tissues, organs and intracellular compartments. Again, pyruvate added to the circulation will need to be converted to lactate prior to entry into cells. The sites of this conversion will be erythrocytes or cytosol of cardiac and skeletal muscle cells. Therefore, provision of pyruvate will only slow delivery of monocarboxylate material for mitochondrial oxidation.
B. Use of Glycerol-lactate Esters as an Energy Source Supplement During Exercise and Recovery:
Recent advances in basic biochemistry and exercise physiology have shown that the formation and removal of lactic acid is an integral part of both digestive and metabolic processes. Further, as lactate is a fuel for the heart (vide supra), it is also a major energy source in working skeletal muscle.
According to the xe2x80x98Glucose Paradoxxe2x80x99 hypothesis (reviewed by Foster, 1984; see also Newgard et al., 1983), dietary carbohydrate courses an indirect route before becoming liver glycogen. It is known that dietary carbohydrate is digested and than enters the portal circulation (i.e., that vein between the small intestine and the liver) largely as glucose.
In contrast to traditional theories which hold that the liver extracts large amounts of portal blood glucose for synthesis of glycogen, it is now believed that portal glucose bypasses the liver and enters the systemic circulation through the hepatic vein. Much of this glucose then reaches the resting musculature, where it is either stored as glycogen or converted into lactic acid. This lactic acid then either diffuses or is transported from the sites of production and reaches the systemic circulation. Much of the circulating lactic acid is removed by the liver.
In the glycogen-depleted liver, lactic-acid becomes the preferred precursor material from which to synthesize glycogen. Because glycogen is paradoxically synthesized by a rather circuitous pathway, the process is alternatively termed the Glucose Paradox, or the Indirect Glucose to Liver Glycogen Pathway.
According to the xe2x80x98Lactate Shuttlexe2x80x9d hypothesis (Brooks, 1985, 1986a, 1986b), 1987, 1998, 1999a, 1999b); lactic acid is an important fuel source for exercise as well as resting and exercise-recovery conditions (FIG. 7). During exercise, active fast-twitch muscles produce lactic acid, which is then available as a fuel for slow-twitch, highly oxidative skeletal muscle fibers (Donovan and Brooks, 1983). This process appears to operate all the time as demonstrated in human subjects exercising at sea level (Bergman et al 1999; Mazzeo et al. 1986; Stanley et al. 1985, 1986, 1988), or high altitude (Brooks et al., 1991, 1992). In fact, based on conclusions conducted on rats (Brooks and Donovan, 1983; Donovan and Brooks, 1983) and humans (Bergman et al., 1999; Brooks, 1992; Stanley et al., 1988), lactate appears to be a more important fuel for muscular exercise than does glucose, especially during sustained exercise and recovery form sustained, exhausting exercise (FIG. 8).
Results of studies conducted by Gladden and associates (1991, 1994) on canine muscles made to contract in situ support observations made on human subjects. The data clearly show that working canine muscles consume and utilize lactate in a concentration-dependent manner.
The oxidation of lactic acid during exercise can be appreciated on both relative and absolute bases. Of the lactic acid produced and removed during exercise, approximately 75% is removed by oxidation and about 20% is converted to glucose (Bergman et al. 1999; Donovan, C. M. and G. A. Brooks, 1983; Stanley et al., 1988, Brooks et al, 1991b, 1992). Of this latter portion, most will ultimately be oxidized also (Brooks, and Donovan, 1983, Brooks et al. 1992). Quantitatively, lactic acid oxidation exceeds glucose oxidation during exercise with 10-25% of the total energy supplied derived from lactic acid oxidation. These findings suggest that it may be desirable to employ lactic acid as a supplement during and/or after exercise.
However, the use of lactic acid as a fuel in the body carries with it potential penalties. Lactic acid accumulation in the muscle is painful and interferes with contraction processes. Further, large amounts of lactic acid in the blood cause pH to fall which is physically and psychologically distressing to the performer. These disadvantages are associated with the hydrogen ion (H+, or proton) which results when lactic acid dissociates in aqueous solutions. For these reasons lactic acid accumulation has long been suspected as a cause of muscle fatigue (Brooks et al., Exercise Physiology: Human Bioenergetics and its Applications, Chapter 33, Mayfield, Mountainview, Third Edition, 2000).
Therefore, it may be advantageous to provide a carbohydrate derived fuel source to an individual engaged in prolonged, strenuous exercise, and it would be more efficacious to provide the carbohydrate energy in the form of a xe2x80x98lactic acid-likexe2x80x99 substance which would provide a more immediate fuel source.
Thus, on the bases of both the xe2x80x98Glucose Paradoxxe2x80x9d and xe2x80x98Lactate Shuttlexe2x80x99 concepts, providing a xe2x80x98lactic acid-likexe2x80x99 material to athletes during exercise and recovery from exercise would also augment the beneficial effects of providing dietary glucose.
This invention relates to a new lactate compound and a method of: (1) providing energy to the heart and skeletal muscles during physical exercise and recovery from exercise, and (2) providing a supplemental energy source to active mammals during exercise and recovery from exercise. The invention is particularly directed to: (1) a method of cardiac and skeletal muscle energy supplementation during and following energy demanding activities, (2) a method of replenishing energy in active individuals, (3) a method of maintaining blood sugar (glucose) in exercising individuals and restoring liver carbohydrate stores (glycogen) during recovery from exercise, and (4) a method of hydrating and rehydrating individuals during exercise and recovery. The inventive method is constructed to benefit: (1) cardiac and skeletal muscle energy resuscitation during and following strenuous exercise, and (2) increase the energy supply and vigor of active individuals. Accordingly, the global objective of this invention is to provide a new and improved lactate compound.
Specific objectives of this invention are, in mammals:
(1) to provide a new and improved method to provide energy to the stressed heart,
(2) to provide a new and improved method to provide energy to stressed skeletal muscle, and
(3) to provide a new and improved method to supply supplemental energy to exercising individuals, and
(4) to facilitate hydration of individuals before, during and after exercise.
To achieve the foregoing objects and in accordance with the purpose of the invention, as embodied and broadly described herein, the novel lactate compound of this invention comprise a glycerol-lactate ester (GLE). Preferably, the ester is in the tri-lactate form, but other lactate-ester forms (di- or mono-lactate esters), as well as glycerol-acetate esters (GAE) will serve similar functions. In a particularly preferred form, the compound is a tri-lacteal ester of the glycerol.
Each objective of the invention can be accomplished in mammals, including, but not limited to, horses, canines, and humans. In a most preferable embodiment, this invention envisions treating humans.
The general form of the compound is: 
where R1, R2, and R3 are selected from lactoyl or acetyl groups.
In a the most preferable embodiment, the composition has the formula: 
The invention is directed to use of the novel lactate compound for intravenous, intracoronary, or oral introduction, most preferably oral. Accordingly, the invention includes methods for providing fluid, energy and electrolytes to physically active persons or those exposed to hot, and hot-humid environments, as well as for the preservation of tissue deprived to oxygen through events including, but not limited to, coronary infarction, stroke, mesenteric infarction, organ transplant (during preservation and intravenously after grafting to the organ), including amputated limbs. The composition can be used on any organ or tissue in the body, including, but not limited to, cardiac muscle, skeletal muscle, or brain.
In accordance with the present invention, in addition to providing an energy source following prolonged and demanding exercise, GLE is presented as a novel method and composition beneficial to a mammal""s fluid, electrolyte and carbohydrate balance during exercise and subsequent recovery are provided.
In one aspect, the invention provides a method of supplying nutritional supplementation to humans and other mammals by means of an aqueous solution of at least one lactic acid salt. This solution is administered in oral dosage form to the host in an amount sufficient to affect the mammal""s fluid, electrolyte or carbohydrate balance during exercise and/or subsequent recovery.
In another aspect, a nutritional supplement is provided comprising an aqueous solution of at least one lactic acid salt in an amount sufficient to affect a mammal""s fluid, electrolyte or carbohydrate balance during exercise and/or subsequent recovery.
In another aspect a nutritional supplement is provided to maintain blood glucose during exercise and restore liver glycogen after exercise.
In other aspects, the present nutritional supplement includes mixtures of organic and inorganic lactic acid salts, lactate polymers, and/or simple complex carbohydrates. Such mixtures containing fructose, glucose polymers and larger polysaccharides for provision of fuel energy via enteral (oral) administration represent a different adaptation of the composition than for cardioplegic administration into the blood.